The hormonal control of amaranthin synthesis in Amaranthus caudatus seedlings

Summary Exogenous gibberellic acid, A₃ (GA₃) inhibits phytochrome mediated betacyanin synthesis in seedlings of Amaranthus caudatus. The growth retardants, -chloroethyl-trimethylammonium chloride (CCC), 'isopropyl-4-(triethylammonium chloride)-5-methylphenyl piperidine carboxylate (AMO 1618) and tributyl-2,4,-dichlorobenzylphosphonium chloride (phosphon D) enhance pigment synthesis. Retardant stimulation of pigment synthesis is overcome by GA₃ application. Besides lowering endogenous GA levels the retardants inhibit protein synthesis by as much as 25%. Retardant inhibition of protein synthesis is not overcome by GA₃. The results suggest that amaranthin synthesis in Amaranthus caudatus can be directly controlled by endogenous GA. GA₃ has no effect on kinin induced dark pigment synthesis. Kinins, however, do not overcome GA₃ inhibition of pigment synthesis in the light.Abbreviations AMO 1618 2, 'isopropyl-4-(triethylammonium chloride)-5-methylphenyl piperidine carboxylate - CCC -chloroethyltrimethylammonium chloride - GA₃ Gibberellic acid, A3 - Phosphon D tributyl-2,4,-dichlorobenzylphosphoninm chloride     

Proton and anion transport at the tonoplast in crassulacean-acid-metabolism plants: specificity of the malate-influx system in Kalanchoë daigremontiana

Tonoplast vesicles were prepared from leaf mesophyll homogenates of the crassulacean-acid-metabolism plant Kalanchoë daigremontiana Hamet et Perrier de la Bâthie to study the effects of anions on ATP- and inorganic-pyrophosphate (PPi)-dependent H⁺ transport. In the presence of gramicidin, substrate hydrolysis by the tonoplast ATPase was characteristically stimulated by chloride and inhibited by nitrate, but was unaffected by malate and a wide range of other organic-acid anions; the PPiase was anion-insensitive. Malate was more effective than chloride both in stimulating ATP- and PPi-dependent vesicle acidification (measured as quinacrine-fluorescence quenching) and in dissipating a pre-existing inside-positive membrane potential (measured as oxonol-V-fluorescence quenching), indicating that malate was more readily transported across the tonoplast. Certain other four-carbon dicarboxylates also supported high rates of vesicle acidification, their order of effectiveness being fumarate malate -succinate > oxalacetate - tartrate; the five-carbon dicarboxylates 2-oxoglutarate and glutarate were also transported, although at lower rates. Experiments with non-naturally occurring anions indicated that the malate transporter was not stereospecific, but that it required the trans-carboxyl configuration for transport. Shorter-chain or longer-chain dicarboxylates were not transported, and neither were monocarboxylates, the amino-acid anions aspartate and glutamate, nor the tricarboxylate isocitrate. The non-permeant anions maleate and tartronate appeared to be competitive inhibitors of malate transport but did not affect chloride transport, indicating that malate and chloride influx at the tonoplast might be mediated by separate transporters.Abbreviations BTP 1,3-bis-[tris(hydroxymethyl)methylamino]-propane - CAM crassulacean acid metabolism - oxonol V bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol - pH transmembrane pH difference - PPi inorganic pyrophosphate - PPiase inorganic pyrophosphatase - quinacrine 6-chloro-9-{[4-(diethylamino)-1-methylbutyl]amino}-2-methoxyacridine dihydrochloride - transmembrane electrical potential difference     

The effect of 2,4-dichlorophenoxyacetic acid and (2-chloroethyl)-trimethylammonium chloride on chlorophyll synthesis in barley leaves

Summary 2.4-dichlorophenoxyacetic acid (2.4-D) and (2-chloroethyl)-trimethylammonium chloride (CCC) inhibit chlorophyll synthesis and protochlorophyllide ₆₅₂ regeneration in 6–8 day old barley leaves whilst having little effect on the rates of protochlorophyll ₆₃₂ synthesis from exogenous -aminelevulinic acid (ALA) and ALA-dehydratase activity. Longer pretreatments with 2.4-D and CCC show it is only after 50 to 60 hr that the rates of P₆₃₂ production from exogenous ALA and ALA-dehydratase activity are affected. Similar response times were obtained for chloramphenicol (CAP). The results indicate that 2.4-D and CCC may act by directly inhibiting specific plastid-protein synthesis similar to CAP. Hence it seems that it is only those proteins (enzymes) having a rapid turnover that are affected first i.e. the enzymes necessary for ALA synthesis in the plastid.Abbreviations used ALA -aminolevulinic acid - CAP chloramphenicol - CCC (2-chloroethyl)-trimethylammonium chloride - 2.4-D 2-4-dichlorophenoxyacetic acid - P₆₅₂ prodochlorophyllide with maximum in-vivo absorption at 652 nm - P₆₈₄ chlorophyllide absorbing at 684 nm - P₆₇₀ chlorophyllide absorbing at 670 nm - P₆₃₂ pigment absorbing at 632 nm synthesised from exogenous ALA - PBG Porphobilinogen P. R. Shewry is in receipt of a Science Research Council Studentship award.     

Photocontrol of gibberellin levels as related to the unrolling of etiolated wheat leaves

Summary The rapid increase in acidic gibberellin-like (GA-like) activity occurring in etiolated wheat (Triticum aestivum L.); leaf tissue in response to red light is phytochrome controlled. The production of some of the GA-like activity appears to possess a similar escapetime from far-red reversibility to leaf unrolling and is sensitive to Amo 1618. Application of these GA-like substances to leaf sections maintained in the dark causes unrolling. Increased GA-like activity can also be detected in non-irradiated portions of partly irradiated leaves.Abbreviations GA gibberellin - Amo 1618 2-isopropyl-4-(trimethylammonium chloride)-5-methylphenyl piperidine carboxylate - CCC -chloroethyltrimethyl ammonium chloride     

Effect of inhibitors of gibberellin biosynthesis on the induction of α-amylase in embryoless caryopses of Hordeum vulgare cv. Himalaya

The induction of -amylase by exogenously supplied gibberellin A₁ (GA₁) and GA₄ in embryoless caryopses of Hordeum vulgare (cv. Himalaya) was determined indirectly by measuring reducing sugars released from the endosperm. The presence of the inhibitors of GA biosynthesis, 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (Amo 1618), Ancymidol, 2-chloroethyl trimethyl ammonium chloride (CCC) or (R,S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,3-triazolyl)pentan-3-ol (PP333) did not inhibit -amylase production by either GA₁ or GA₄.Abbreviations Amo-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride - CCC 2-chloroethyl trimethyl ammonium chloride - cv. cultivar - GA gibberellin - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - PP333 (R,S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,3-triazolyl) pentan-3-01     

Active chloride transport in rabbit thick ascending limb of Henle's loop and elasmobranch rectal gland: chloride fluxes in isolated plasma membranes

Summary To investigate directly whether a sodium-potassium-chloride cotransport system is operating in the mammalian thick ascending limb of Henle's loop (TALH) and in the elasmobranch rectal gland, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and from rectal glands ofSqualus acanthias, and chloride uptake was measured by a rapid filtration technique. Chloride uptake into TALH vesicles in the presence of a 25 mM Na₂SO₄, 25 mM K₂SO₄ gradient reached 70% of equilibrium at 2.5 min. In the presence of both sodium and potassium, the 15 s chloride uptake was inhibited 35% by 1 mM bumetanide. When either sodium or potassium was removed from the incubation medium, chloride uptake decreased to the level observed in the presence of 1 mM bumetanide. 0.5 mM SITS had no effect on chloride uptake by the plasma membrane vesicles. This sodium and potassium dependent, bumetanide sensitive chloride uptake was also observed under tracer exchange conditions. Chloride uptake into rectal gland plasma membrane vesicles in the presence of a 50 mM Na₂SO₄, 50 mM K₂SO₄ gradient reached 80% of equilibrium at 2.5 min. 1 mM bumetanide inhibited the 15 s uptake of chloride by 34% and removal of either sodium or potassium from the incubation medium reduced chloride uptake to the level observed in the presence of bumetanide under both gradient and tracer exchange conditions. These studies provide additional support for the hypothesis that a sodium-potassium-chloride cotransport system is operating in these epithelia.Abbreviations SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid - TALH thick ascending limb of Henle's loop     

Modulation of adrenal cell functions by cadmium salts. 1. Cadmium chloride effects on basal and ACTH-stimulated steroidogenesis

Cultured Y-1 mouse adrenal tumor cells, which secrete 20--hydroxy-4-pregnen-3-one (20-DHP), were used to investigate the acute nonlethal effects of incremental cadmium chloride (CdCl₂) concentrations on basal and maximally stimulated steroid secretion. In addition, cumulative CdCl₂ effects during 4-hr incubations, effect reversibility, and viability were determined. Cells were incubated in 1 ml serum-free Eagle's Minimal Essential Medium (FMEM) with or without 0.5 IU (ca. 1.5 M) adrenocorticotropin (ACTH) in the presence or absence of CdCl₂. Following incubation, cell viability was quantitated using trypan blue exclusion. The 20-DHP secreted into the experimental incubation medium was measured by radioimmunoassay. CdCl₂ levels of 10.0 g/ml or greater significantly inhibited basal 30 min steroid secretion in a dose-dependent manner; ACTH-stimulated steroid secretion was significantly inhibited by levels 5.0 g/ml or greater. At least 80% of all control and stimulated cells in the presence or absence of cadmium ions excluded trypan blue. The reduction in ACTH-stimulated steroid secretion was greater than the reduction in basal steroid secretion at any cadmium concentration level. The CdCl₂ concentration that reduced stimulated steroid hormone secretion by 50% (IC₅₀) was 45.0 g/ml. Exposing Y-1 cells to either 5.0, 10.0, 45.0 or 500.0 g CdCl₂/ml FMEM for periods ranging from 0.5 to 4 hr inhibited ACTH-stimulated steroid secretion in a time-dependent manner. After 30 min exposure to 10.0, 45.0 or 500.0 g CdCl₂/ml FMEM with or without ACTH, cadmium inhibition was irreversible. When 5.0 g CdCl₂/ml was used, basal and stimulated inhibition was reversible by reincubating in medium containing ACTH alone. The relatively greater cadmium effects on ACTH stimulated steroidogenesis might suggest that cadmium modulated the rate-limited transducing system between the ACTH plasma membrane receptor complex and cholesterol side-chain cleaving mitochondrial enzymes. However, cadmium influences on basal secretion indicated effects on the non-rate-limited steroidogenic pathway.Abbreviations ACTH adrenocorticotropin - ANOVA analysis of variance - CdCl₂ cadmium chloride - Ci Curie - DNA deoxyribonucleic acid - FMEM serum-free Eagle's Minimum Essential Medium - HEPES N-2-hydroxyethylpiperazine-N-1,2-ethanesulfonic acid - IC₅₀ concentration inhibiting stimulated steroid secretion by 50% - IU international unit - MEM Eagle's Minimum Essential Medium - RIA radioimmunoassay - RNA ribonucleic acid - SEM standard error of the mean - SMEM serum-containing Eagle's Minimum Essential Medium - 20-DHP 20--hydroxy-4-pregnen-3-one     

Chloride transport across the integumentary epithelium ofManduca sexta (Lepidoptera: Sphingidae)

Summary Moulting fluid ofManduca sexta contains high concentrations of potassium and bicarbonate (100 mM) and low concentrations of chloride (5 mM). This fluid begins to disappear from the exuvial space approximately 9–10 h before the actual shedding of the integument. During this time, the integument can be isolated in an Ussing cell and electrical properties measured in vitro. In a normal 32 mM KHCO₃ saline, potential difference (PD) is around 10 mV, exuvial side positive, and short-circuit current (SCC) is 15–20 A cm^–2. Substitution of chloride slightly reduces both PD and SCC, although resistance does not change significantly. Measurement of chloride transport in the absence of K⁺ indicates that 100% of the SCC can be accounted for by the net chloride flux (2 A cm^–2). TheK _m andJ _max for transepithelial chloride transport are 14 mM and 0.1 Eq cm^–2 h^–1. Bilateral potassium addition stimulates chloride transport, doubling net chloride flux as potassium concentration increases from 2 to 5 mM. Chloride net flux is not inhibited by the presence of furosemide (1 mM), nor in HCO ₃ ^– -free saline by thiocyanate (1 or 10 mM) or acetazolamide (0.1 mM), but is inhibited by 100% N₂. The pattern of chloride transport inM. sexta is similar to that previously reported for the rectum of locusts. As chloride is normally at low concentrations in the moulting fluid, it is suggested that this transport system acts to maintain low intracellular concentrations which may be necessary for enzymatic functions in the epidermal cells and has little importance in fluid transport.Abbreviations PD potential difference - PPI pharate pupal integument - SCC short circuit current In the time since this research was performed, A.M. Jungreis passed away. He will be missed by his friends and colleagues     

Interactive effects of GAs,CKs and growth retardants on the germination of celery seeds

The germination of whole seeds of celery (Apium graveolens L.) was inhibited by paclobutrazol, ancymidol and lower concentrations of uniconazole. The growth retardants daminozide, AMO 1618 and chlormequat chloride inhibited the germination of cut seeds only, indicating that the seed coat prevents the penetration of these compounds at the examined concentrations. Application of a mixture of the gibberellins A4 and A7 (GA 4/7) reversed the inhibition of all the examined growth retardants. Cytokinins (artificial or natural) had no effect when applied alone and did not interact with GA4/7 in the light. However, in the dark the cytokinins at some concentrations and GA4/7 had a synergistic effect in reversing the inhibition caused by growth retardants to whole seeds or in promoting the germination of whole seeds. It is therefore suggested that the major effect on seed of exogenous cytokinins when applied together with GA's is to increase the uptake of gibberellins by the seeds.Abbreviations AMO 1618 (2 isopropyl-5-methyl-4-trimethylammonium chloride-phenyl-1-Piperidinium-carboxylate - ancymidol -cyclopropyl-[4-methoxyphenyl]-5-pyrimidinemeth anol] - chlormequat chloride 2-chloroethyltrimethylommonium chloride - daminozide succinin acid 2,2-diamethyl hydrazide - paclobutrazol [2 RS, 3 RS]-1-(4-chlorophenyl)-4,4-dimethyl-2-(1 H-1,2,4-triazol-1-yl) pentan-3-ol - uniconazole (E)-1-(P-chlorophenyl)-4,4-dimethyl-2-(1,2,4 triazol-1-yl)-1 penten-3-ol     

Gibberellin involvement in dormancy-break and germination of seeds of celery (Apium graveolens L.)

The temperature-dependent, primary dormancy of cv. Florida 683 celery seeds in darkness was partially broken by a 30 min light exposure on the third day of incubation at 20–22°C, resulting in c 50 percent germination after 20 days. This light stimulation was negated by including different inhibitors of gibberellin biosynthesis in the incubation medium. Subsequent addition of a solution of the gibberellins A₄ and A₇ or of the gibberellin-active compound (1-3-chlorophthalimido)-cyclohexane carboxamide (AC94,377) overcame the inhibitory effects on germination of these GA-biosynthesis inhibitors. It is suggested that light stimulates the biosynthesis of gibberellins which are essential for dormancy-break in celery seeds and that this biosynthesis is either directly or indirectly controlled through phytochrome.Abbreviations AC94,377 1-(3-chlorophthalimido)-cyclohexane carboxamide; ancymidol, -cyclopropyl--(4-methoxyphenyl)-5-pyrimidinemethanol - AMO1618 N,N,N-2-tetramethyl-5-(1-methyl ethyl)-4-(1-piperidinylcarbonyl)oxy-benzenaminium chloride - BTS44584 S-2,5-dimethyl-4-pentamethylenecarbamoyloxyphenyl-SS-dimethyl sulphonium - P toluenesulphonate; chlormequat chloride, 2-chloroethyltrimethylammonium chloride; daminozide - N dimethylaminoscuccinamic acid; paclobutrazol, (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl pentan-3-ol)     

Agreement of blood spot card measurements of vitamin D levels with serum, whole blood specimen types and a dietary recall instrument

Background

The ability to measure 25-hydroxyvitamin D (25OHD) levels from blood spot cards can simplify sample collection versus samples obtained by venipuncture, particularly in populations in whom it is difficult to draw blood. We sought to validate the use of blood spot samples for the measurement of 25OHD compared to serum or whole blood samples and correlate the measured levels with intake estimated from dietary recall.

Methods

Utilizing 109 biological mothers of infants enrolled in the Tennessee Children''s Respiratory Initiative cohort, we measured 25OHD levels through highly selective liquid chromatography–tandem mass spectrometry on samples from blood spot cards, serum, and whole blood collected at enrollment. Dietary questionnaires (n = 65) were used to assess 25OHD intake by dietary recall. Sample collection measures were assessed for agreement and 25OHD levels for association with dietary 25OHD intake.

Results

The mean absolute differences (95%CI) in 25OHD levels measured between whole blood and blood spot (n = 50 pairs) or serum and blood spot (n = 20) were 3.2 (95%CI:1.6, 4.8) ng/ml and 1.5 (95%CI:−0.5,3.4) ng/mL. Intake by dietary recall was marginally associated with 25OHD levels after adjustment for current smoking and race in linear regression.

Discussion

25OHD levels determined by mass spectrometry from blood spot cards, serum and whole blood show relatively good agreement, although 25OHD levels are slightly lower when measured by blood spot cards. Blood spot samples are a less invasive means of obtaining 25OHD measurements, particularly in large population-based samples, or among children when venipuncture may decrease study participation.     

The effect of the polar moiety of lipids on the ion permeability of bilayer membranes

Summary Bilayer membranes were prepared with the negatively charged lipids phosphatidylglycerol and diphosphatidylglycerol, the positively charged lipid lysyl phosphatidylglycerol, the zwitterionic lipid phosphatidylethanolamine, and an uncharged glycolipid, diglucosyldiglyceride, all isolated from gram-positive bacteria. Bilayer membranes of all these lipids manifested specific resistances of 10⁷ to 10⁹ cm² and capacitances of 0.3 to 0.4 F cm^–2. The membrane potentials of these bilayers were measured as a function of the sodium chloride, potassium chloride, and hydrogen chloride transmembrane concentration gradients (0.01 to 0.10m) and were found to be linear with the logarithm of the salt activity gradients. Membranes made from lysyl phosphatidylglycerol (one net positive charge) were almost completely chloride selective, whereas membranes from phosphatidylglycerol and diphosphatidylglycerol (one and two net negative charges, respectively) were highly cation selective. Membranes prepared with either diglucosyldiglyceride or phosphatidylethanolamine showed only slight cation selectivity. These findings indicate that the charge on the polar head group of membrane lipids plays an important role in controlling the ion-selective permeability of the bilayer.     

The action of plant growth retardants on terpenoid biosynthesis

Summary The plant growth retardants (2-chloroethyl)-trimethylammonium chloride (CCC) and 2-isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl-piperidine-1-carboxylate (AMO-1618) inhibit gibberellic-acid biosynthesis inFusarium moniliforme at the cyclisation of geranylgeraniol to (-)-kaurene, causing an accumulation of geranylgeraniol. The two inhibitors have no effect on the biosynthesis of ergosterol inF. moniliforme or sitosterol in barley seedlings.     

The reduction of tetrazolium salts by plant mitochondria

Summary Data has been obtained concerning the reduction of tetrazolium salts by mitochondria isolated from Jerusalem artichoke tubers with succinate as the substrate using a direct recording spectrophotometric method of assay. ATP was found to increase the rate of reduction of the tetrazolium salts, this being independent of the effect ATP had on the rate of oxygen uptake. The magnitude of the stimulation by ATP depended on the concentration of tetrazolium salts present and under certain circumstances was suppressed by the addition of azide and cyanide. The sites at which the tetrazolium salts were reduced along the electron transport chain were investigated. The role of ATP has been discussed in relation to the mechanism of tetrazolium reduction.Abbreviations TTC 2,3,5-triphenyl-2,1,3,4-tetrazolium chloride - BT 5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-diphenylene)-ditetrazolium chloride - NT 2,2,5,5-tetraphenyl-3,3-(p-diphenylene)-ditetrazolium chloride - MTT 3-(4,5-dimethyl thiozolyl-2)-2,5-diphenyl tetrazolium bromide - INT 2-(p-iodophenyl)-3-p-dinitrophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride - NBT 2,2-dinitrophenyl-5,5-diphenyl-3,3-dimethoxy-4,4-diphenylene)-ditetrazolium chloride - TNBT 2,2-5,5-tetra-p-nitrophenyl-3,3-dimethoxy-4,4-diphenylene) ditetrazolium chloride     

The effect of growth retardants on anthocyanin production in carrot cell suspension cultures

The effect of growth retardants on anthocyanin production was studied in wild carrot (Daucus carota) cell suspension cultures. Paclobutrazol [(2RS,3RS) — 1 — (4-chlorophenyl) — 4,4 —dimethyl-2-(1,2,4-triazol-1-yl) pentan-3-ol], uniconazole [(E)-1-(4-chlorophenyl-4,4 —) dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol], tetcyclacis [5-(4-chloro-phenyl) -3,4,5,9,10-pentaaza-tetracyclo-5, 4, 10^2,6, O^8,11 — dodeca-3, 9-diene], ancymidol [-cyclopropyl — 4 — methoxy-(pyrimidine-5-yl)benzyl alcohol] and CCC (2-chloro-ethyltrimethylammonium chloride) increased anthocyanin accumulation. AMO-1618 [(2-isopropyl-5-methyl-4-trimethyl-ammonium-chloride)-phenyl-1-piperidinium carboxylate] did not increase anthocyanin accumulation in the first passage but did increase it during the second passage on medium for improved anthocyanin accumulation. Prohexadione (3,5-dioxo-4-propionylcyclohexane carboxylic acid) decreased anthocyanin accumulation by 10%–12.5%.The inhibitory effect of gibberellin on anthocyanin accumulation was reversed by paclobutrazol. Paclobutrazol together with 10^–6M GA₃ increased anthocyanin level from 33% of control in GA₃ treated cell suspension to 76%. These results are consistent growth retardants increasing anthocyanin accumulation in carrot cell suspension cultures by inhibiting gibberellin biosynthesis.     

The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures

Background

Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, β-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6^th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, β-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells.

Results

Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 μg/100 g) were found in cells treated with 1.0 mM CdCl₂ and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl₂ gradually increased during the culture period and the highest values of α, β and γ tocopherols (145.61, 25.52 and 18.56 μg/100 g) were detected in the cell cultures collected at day 6.

Conclusions

As a conclusion, secondary metabolite contents were increased by cadmium chloride application and sampling time, while dry cell weights was reduced by cadmium chloride treatments.     

Reversing the inhibitory effect of paclobutrazol on seed germination of Amaranthus paniculatus by GA3, ethephon or ACC

The germination of Amaranthus paniculatus seeds was inhibited by applying paclobutrazol, a specific inhibitor of gibberellin biosynthesis. This inhibition was markedly counteracted by gibberellin A₃ (GA₃), suggesting that endogenous gibberellins are required for germination in this species. The inhibitory effect of paclobutrazol was also overcome by ethephon (2-chloroethylphosphonic acid) or the precursor of ethylene biosynthesis, ACC (1-aminocyclopropane-l-carboxylic acid). Thus the physiological effect of gibberellin can be mimicked by ethylene released from ethephon or synthesised from exogenous ACC. It is suggested, that endogenous gibberellins are involved in germination of Amaranthus paniculatus seeds and that action of GA₃ can be substituted by ethylene.Abbreviations ACC 1-aminocyclopropane-l-carboxylic acid - AMO-1618 (2-isopropyl-5methyl-4-trimethylammoniumchloride)-phenyl-l-piperidinium-carboxylate - ancymidol -cyclopropyl--(4-methoxyphenyl)-5-pyrimidine methanol - chloromequat chloride (2-chloroethyl)trimethylammoniumchloride - ethephon 2-chloroethylphosphonic acid - GA gibberellin A₃ - paclobutrazol (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-lyl)pentan-3-ol - Phosphon D 2,4,dichlorobenzyl-tributhylphosphoniumchloride - tetcyclacis 5,(4-chlorophenyl)-3,4,5,9,10-pentaaza-tetracyclo)5,4,1,0,Z,6,0^8,11 dodeca-3,9-diene     

Biopolymer–surfactant interaction. I. Kinetics of binding of cetyltrimethyl ammonium bromide with carboxymethyl cellulose (Na Salt)

The kinetics of binding of the cationic surfactant cetyltrimethyl ammonium bromide with the Na salt of carboxymethyl cellulose was studied by the electrometric method using cetyltrimetlyl ammonium⁺ (CTA⁺) ion-selective polyvinyl chloride membrane electrode. The binding process followed the first-order kinetics and occurred in three stages. Its affinity increased with increasing CTA bromide concentration and decreased with ionic strength. The activation process comprised moderate E and ΔH^≠ and negative ΔS^≠ for all three stages with a ΔH^≠ < TδS^≠ trend proving it to be entropy controlled. The ΔG^≠ values followed the trend ΔG < ΔG < ΔG (in accordance with k₁ > k₂ > k₃). The enthalpies (ΔH^≠) and entropies (ΔS^≠) of activation followed a systematic and interdependent trend. The multiple-stage binding kinetics is grossly comparable with the kinetics of binding of proteins to solid surfaces. © 1995 John Wiley & Sons, Inc.     

Nitro-blue tetrazolium: A specific stain for photosynthetic activity in protoplasts

A qualitative assay for the detection of photosynthetic activity in protoplasts is described. Leaf protoplasts from atrazine resistant and susceptible biotypes of Brassica napus suspended in medium with 0.01% nitro-blue tetrazolium and 10 to 100 µM atrazine showed clear differences in staining. Staining was detectable with 0.001% fluorescein-labelled tetrazolium which, unlike nitro-blue tetrazolium, did not cause collapse of the protoplasts.Abbreviations atrazine 2-chloro-4-(ethylamino)-6(isopropyl-amino)-s-triazine - DCPIP dichlorophenolindophenol - fluorescein-labelled tetrazolium 2,5-bis-(4-nitrophenyl)-3-(5-fluoresceinyl)-2H-tetrazolium chloride - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino)ethanesulfonic acid - nitro-blue tetrazolium 2,2,5,5-tetraphenyl-(3,3-dimethoxy-4,4-biphenylene)-ditetrazolium chloride - PS 0.6M sorbitol + 10mM NaHCO3 + 50mM Hepes, pH 7.6 - SM 0.5M sorbitol + 10mM Mes, pH 5.8 This work has been submitted by D. R. in partial fulfillment of the requirement for the Ph.D. degree     

Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L.

Mesophyll protoplasts were isolated from unhardened and cold-acclimated leaves of Valerianella locusta L. and subjected to freeze-thaw treatment. To evaluate the extent and course of freezing injury, photosynthetic reactions of whole protoplasts and of free thylakoid membranes, liberated from protoplasts by osmotic lysis, were measured. In addition, the integrity of the protoplasts was determined by microscopy. The results reveal an increased frost tolerance of protoplasts isolated from acclimated leaves with respect to all parameters measured. CO₂-dependent O₂ evolution (representing net photosynthetic CO₂ fixation of protoplasts) was the most freezing-sensitive reaction; its inhibition due to freeze-thaw treatment of protoplasts was neither correlated with disintegration of the plasma membrane, nor was it initiated by inactivation of the thylakoid membranes. The frost-induced decline of protoplast integrity was not closely correlated to thylakoid damage either. Freezing injury of the thylakoid membranes was manifested by inhibition of photosynthetic electron transport and photophosphorylation. Both photosystems were affected by freezing and thawing with strongest inhibition occurring in the water-oxidation system or at the oxidizing site of photosystem II. Photophosphorylation responded more sensitively to freezing stress than electron transport, although uncoupling (increased permeability of the thylakoid membranes to protons) was not a conspicuous effect. The data are discussed in relation to freezing injury in leaves and seem to indicate that frost damage in vivo is initiated at multiple sites.Abbreviations Chl chlorphyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - Hepes 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - MES 2-(N-morpholino)-ethanesulfonic acid - PS I photosystem I - PS II photosystem II